Methyltransferase-like 21c methylates and stabilizes the heat shock protein Hspa8 in type I myofibers in mice.

Protein methyltransferases mediate posttranslational modifications of both histone and nonhistone proteins. Whereas histone methylation is well-known to regulate gene expression, the biological significance of nonhistone methylation is poorly understood. Methyltransferase-like 21c (Mettl21c) is a newly classified nonhistone lysine methyltransferase whose in vivo function has remained elusive. Using a Mettl21cLacZ knockin mouse model, we show here that Mettl21c expression is absent during myogenesis and restricted to mature type I (slow) myofibers in the muscle. Using co-immunoprecipitation, MS, and methylation assays, we demonstrate that Mettl21c trimethylates heat shock protein 8 (Hspa8) at Lys-561 to enhance its stability. As such, Mettl21c knockout reduced Hspa8 trimethylation and protein levels in slow muscles, and Mettl21c overexpression in myoblasts increased Hspa8 trimethylation and protein levels. We further show that Mettl21c-mediated stabilization of Hspa8 enhances its function in chaperone-mediated autophagy, leading to degradation of client proteins such as the transcription factors myocyte enhancer factor 2A (Mef2A) and Mef2D. In contrast, Mettl21c knockout increased Mef2 protein levels in slow muscles. These results identify Hspa8 as a Mettl21c substrate and reveal that nonhistone methylation has a physiological function in protein stabilization.

Methyltransferase-like 21e inhibits 26S proteasome activity to facilitate hypertrophy of type IIb myofibers.

Skeletal muscles contain heterogeneous myofibers that are different in size and contractile speed, with type IIb myofiber being the largest and fastest. Here, we identify methyltransferase-like 21e (Mettl21e), a member of newly classified nonhistone methyltransferases, as a gene enriched in type IIb myofibers. The expression of Mettl21e was strikingly up-regulated in hypertrophic muscles and during myogenic differentiation in vitro and in vivo. Knockdown (KD) of Mettl21e led to atrophy of cultured myotubes, and targeted mutation of Mettl21e in mice reduced the size of IIb myofibers without affecting the composition of myofiber types. Mass spectrometry and methyltransferase assay revealed that Mettl21e methylated valosin-containing protein (Vcp/p97), a key component of the ubiquitin-proteasome system. KD or knockout of Mettl21e resulted in elevated 26S proteasome activity, and inhibition of proteasome activity prevented atrophy of Mettl21e KD myotubes. These results demonstrate that Mettl21e functions to maintain myofiber size through inhibiting proteasome-mediated protein degradation.-Wang, C., Zhang, B., Ratliff, A. C., Arrington, J., Chen, J., Xiong, Y., Yue, F., Nie, Y., Hu, K., Jin, W., Tao, W. A., Hrycyna, C. A., Sun, X., Kuang, S. Methyltransferase-like 21e inhibits 26S proteasome activity to facilitate hypertrophy of type IIb myofibers.

AIM2 inflammasome is activated by pharmacological disruption of nuclear envelope integrity.

Inflammasomes are critical sensors that convey cellular stress and pathogen presence to the immune system by activating inflammatory caspases and cytokines such as IL-1ß. The nature of endogenous stress signals that activate inflammasomes remains unclear. Here we show that an inhibitor of the HIV aspartyl protease, Nelfinavir, triggers inflammasome formation and elicits an IL-1R-dependent inflammation in mice. We found that Nelfinavir impaired the maturation of lamin A, a structural component of the nuclear envelope, thereby promoting the release of DNA in the cytosol. Moreover, deficiency of the cytosolic DNA-sensor AIM2 impaired Nelfinavir-mediated inflammasome activation. These findings identify a pharmacologic activator of inflammasome and demonstrate the role of AIM2 in detecting endogenous DNA release upon perturbation of nuclear envelope integrity.

a-Factor Analogues Containing Alkyne- and Azide-Functionalized Isoprenoids Are Efficiently Enzymatically Processed and Retain Wild-Type Bioactivity.

Protein prenylation is a post-translational modification that involves the addition of one or two isoprenoid groups to the C-terminus of selected proteins using either farnesyl diphosphate or geranylgeranyl diphosphate. Three crucial enzymatic steps are involved in the processing of prenylated proteins to yield the final mature product. The farnesylated dodecapeptide, a-factor, is particularly useful for studies of protein prenylation because it requires the identical three-step process to generate the same C-terminal farnesylated cysteine methyl ester substructure present in larger farnesylated proteins. Recently, several groups have developed isoprenoid analogs bearing azide and alkyne groups that can be used in metabolic labeling experiments. Those compounds have proven useful for profiling prenylated proteins and also show great promise as tools to study how the levels of prenylated proteins vary in different disease models. Herein, we describe the preparation and use of prenylated a-factor analogs, and precursor peptides, to investigate two key questions. First, a-factor analogues containing modified isoprenoids were prepared to evaluate whether the non-natural lipid group interferes with the biological activity of the a-factor. Second, a-factor-derived precursor peptides were synthesized to evaluate whether they can be efficiently processed by the yeast proteases Rce1 and Ste24 as well as the yeast methyltransferase Ste14 to yield mature a-factor analogues. Taken together, the results reported here indicate that metabolic labeling experiments with azide- and alkyne-functionalized isoprenoids can yield prenylated products that are fully processed and biologically functional. Overall, these observations suggest that the isoprenoids studied here that incorporate bio-orthogonal functionality can be used in metabolic labeling experiments without concern that they will induce undesired physiological changes that may complicate data interpretation.

Dual Modulation of Human P-Glycoprotein and ABCG2 with Prodrug Dimers of the Atypical Antipsychotic Agent Paliperidone in a Model of the Blood-Brain Barrier.

Many atypical antipsychotic drugs currently prescribed for the treatment of schizophrenia have limited brain penetration due to the efflux activity of ATP-binding cassette (ABC) transporters at the blood-brain barrier (BBB), including P-glycoprotein (P-gp) and ABCG2. Herein, we describe the design and synthesis of the first class of homodimeric prodrug dual inhibitors of P-gp and ABCG2. These inhibitors are based on the structure of the atypical antipsychotic drug paliperidone (Pal), a transport substrate for both transporters. We synthesized and characterized a small library of homodimeric bivalent Pal inhibitors that contain a variety of tethers joining the two monomers via ester linkages. The majority of our compounds were low micromolar to sub-micromolar inhibitors of both P-gp and ABCG2 in cells overexpressing these transporters and in immortalized human hCMEC/D3 cells that are derived from the BBB. Our most potent dual inhibitor also contained an internal disulfide bond in the tether (Pal-8SS) that allowed for rapid reversion to monomer in the presence of reducing agents or plasma esterases. To increase stability against these esterases, we further engineered Pal-8SS to contain two hindering methyl groups alpha to the carbonyl of the ester moiety within the tether. The resulting dimer, Pal-8SSMe, was also a potent dual inhibitor that remained susceptible to reducing conditions but was more resistant to breakdown in human plasma. Importantly, Pal-8SSMe both accumulated and subsequently reverted to the therapeutic Pal monomer in the reducing environment of BBB cells. Thus, these molecules serve two purposes, acting as both inhibitors of P-gp and ABCG2 at the BBB and as prodrugs, effectively delivering therapies to the brain that would otherwise be precluded.

Dual inhibitors of the human blood-brain barrier drug efflux transporters P-glycoprotein and ABCG2 based on the antiviral azidothymidine.

The brain provides a sanctuary site for HIV due, in part, to poor penetration of antiretroviral agents at the blood-brain barrier. This lack of penetration is partially attributed to drug efflux transporters such as P-glycoprotein (P-gp) and ABCG2. Inhibition of both ABCG2 and P-gp is critical for enhancing drug accumulation into the brain. In this work, we have developed a class of homodimers based on the HIV reverse transcriptase inhibitor azidothymidine (AZT) that effectively inhibits P-gp and ABCG2. These agents block transporter mediated efflux of the P-gp substrate calcein-AM and the ABCG2 substrate mitoxantrone. The homodimers function by interacting with the transporter drug binding sites as demonstrated by competition studies with the photo-affinity agent and P-gp/ABCG2 substrate [125I]iodoarylazidoprazosin. As such, these dual inhibitors of both efflux transporters provide a model for the future development of delivery vehicles for antiretroviral agents to the brain.

Human ZMPSTE24 disease mutations: residual proteolytic activity correlates with disease severity.

The zinc metalloprotease ZMPSTE24 plays a critical role in nuclear lamin biology by cleaving the prenylated and carboxylmethylated 15-amino acid tail from the C-terminus of prelamin A to yield mature lamin A. A defect in this proteolytic event, caused by a mutation in the lamin A gene (LMNA) that eliminates the ZMPSTE24 cleavage site, underlies the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS). Likewise, mutations in the ZMPSTE24 gene that result in decreased enzyme function cause a spectrum of diseases that share certain features of premature aging. Twenty human ZMPSTE24 alleles have been identified that are associated with three disease categories of increasing severity: mandibuloacral dysplasia type B (MAD-B), severe progeria (atypical 'HGPS') and restrictive dermopathy (RD). To determine whether a correlation exists between decreasing ZMPSTE24 protease activity and increasing disease severity, we expressed mutant alleles of ZMPSTE24 in yeast and optimized in vivo yeast mating assays to directly compare the activity of alleles associated with each disease category. We also measured the activity of yeast crude membranes containing the ZMPSTE24 mutant proteins in vitro. We determined that, in general, the residual activity of ZMPSTE24 patient alleles correlates with disease severity. Complete loss-of-function alleles are associated with RD, whereas retention of partial, measureable activity results in MAD-B or severe progeria. Importantly, our assays can discriminate small differences in activity among the mutants, confirming that the methods presented here will be useful for characterizing any new ZMPSTE24 mutations that are discovered.

Diazirine-containing photoactivatable isoprenoid: synthesis and application in studies with isoprenylcysteine carboxyl methyltransferase.

Photoaffinity labeling is a useful technique employed to identify protein-ligand and protein-protein noncovalent interactions. Photolabeling experiments have been particularly informative for probing membrane-bound proteins where structural information is difficult to obtain. The most widely used classes of photoactive functionalities include aryl azides, diazocarbonyls, diazirines, and benzophenones. Diazirines are intrinsically smaller than benzophenones and generate carbenes upon photolysis that react with a broader range of amino acid side chains compared with the benzophenone-derived diradical; this makes diazirines potentially more general photoaffinity-labeling agents. In this article, we describe the development and application of a new isoprenoid analogue containing a diazirine moiety that was prepared in six steps and incorporated into an a-factor-derived peptide produced via solid-phase synthesis. In addition to the diazirine moiety, fluorescein and biotin groups were also incorporated into the peptide to aid in the detection and enrichment of photo-cross-linked products. This multifuctional diazirine-containing peptide was a substrate for Ste14p, the yeast homologue of the potential anticancer target Icmt, with K(m) (6.6 µM) and V(max) (947 pmol min(-1) mg(-1)) values comparable or better than a-factor peptides functionalized with benzophenone-based isoprenoids. Photo-cross-linking experiments demonstrated that the diazirine probe photo-cross-linked to Ste14p with observably higher efficiency than benzophenone-containing a-factor peptides.

Inhibitors of protein geranylgeranyltransferase-I lead to prelamin A accumulation in cells by inhibiting ZMPSTE24.

Protein farnesyltransferase (FTase) inhibitors, generally called "FTIs," block the farnesylation of prelamin A, inhibiting the biogenesis of mature lamin A and leading to an accumulation of prelamin A within cells. A recent report found that a GGTI, an inhibitor of protein geranylgeranyltransferase-I (GGTase-I), caused an exaggerated accumulation of prelamin A in the presence of low amounts of an FTI. This finding was interpreted as indicating that prelamin A can be alternately prenylated by GGTase-I and that inhibiting both protein prenyltransferases leads to more prelamin A accumulation than blocking FTase alone. Here, we tested an alternative hypothesis-GGTIs are not specific for GGTase-I, and they lead to prelamin A accumulation by inhibiting ZMPSTE24 (a zinc metalloprotease that converts farnesyl-prelamin A to mature lamin A). In our studies, commonly used GGTIs caused prelamin A accumulation in human fibroblasts, but the prelamin A in GGTI-treated cells exhibited a more rapid electrophoretic mobility than prelamin A from FTI-treated cells. The latter finding suggested that the prelamin A in GGTI-treated cells might be farnesylated (which would be consistent with the notion that GGTIs inhibit ZMPSTE24). Indeed, metabolic labeling studies revealed that the prelamin A in GGTI-treated fibroblasts is farnesylated. Moreover, biochemical assays of ZMPSTE24 activity showed that ZMPSTE24 is potently inhibited by a GGTI. Our studies show that GGTIs inhibit ZMPSTE24, leading to an accumulation of farnesyl-prelamin A. Thus, caution is required when interpreting the effects of GGTIs on prelamin A processing.

Toward eradicating HIV reservoirs in the brain: inhibiting P-glycoprotein at the blood-brain barrier with prodrug abacavir dimers.

Eradication of HIV reservoirs in the brain necessitates penetration of antiviral agents across the blood-brain barrier (BBB), a process limited by drug efflux proteins such as P-glycoprotein (P-gp) at the membrane of brain capillary endothelial cells. We present an innovative chemical strategy toward the goal of therapeutic brain penetration of the P-gp substrate and antiviral agent abacavir, in conjunction with a traceless tether. Dimeric prodrugs of abacavir were designed to have two functions: inhibit P-gp efflux at the BBB and revert to monomeric therapeutic within cellular reducing environments. The prodrug dimers are potent P-gp inhibitors in cell culture and in a brain capillary model of the BBB. Significantly, these agents demonstrate anti-HIV activity in two T-cell-based HIV assays, a result that is linked to cellular reversion of the prodrug to abacavir. This strategy represents a platform technology that may be applied to other therapies with limited brain penetration due to P-glycoprotein.

Evaluation of substrate and inhibitor binding to yeast and human isoprenylcysteine carboxyl methyltransferases (Icmts) using biotinylated benzophenone-containing photoaffinity probes.

Isoprenylcysteine carboxyl methyltransferases (Icmts) are a class of integral membrane protein methyltransferases localized to the endoplasmic reticulum (ER) membrane in eukaryotes. The Icmts from human (hIcmt) and Saccharomyces cerevisiae (Ste14p) catalyze the α-carboxyl methyl esterification step in the post-translational processing of CaaX proteins, including the yeast a-factor mating pheromones and both human and yeast Ras proteins. Herein, we evaluated synthetic analogs of two well-characterized Icmt substrates, N-acetyl-S-farnesyl-L-cysteine (AFC) and the yeast a-factor peptide mating pheromone, that contain photoactive benzophenone moieties in either the lipid or peptide portion of the molecule. The AFC based-compounds were substrates for both hIcmt and Ste14p, whereas the a-factor analogs were only substrates for Ste14p. However, the a-factor analogs were found to be micromolar inhibitors of hIcmt. Together, these data suggest that the Icmt substrate binding site is dependent upon features in both the isoprenyl moiety and upstream amino acid composition. Furthermore, these data suggest that hIcmt and Ste14p have overlapping, yet distinct, substrate specificities. Photocrosslinking and neutravidin-agarose capture experiments with these analogs revealed that both hIcmt and Ste14p were specifically photolabeled to varying degrees with all of the compounds tested. Our data suggest that these analogs will be useful for the future identification of the Icmt substrate binding sites.

Click chemistry-derived bivalent quinine inhibitors of P-glycoprotein-mediated cellular efflux.

P-glycoprotein (P-gp) effluxes a diverse set of drug substrates out of cells in an ATP dependent manner, thereby limiting the effective accumulation of therapeutic agents. Herein we demonstrate the use of click chemistry to rapidly generate bivalent quinine dimers, containing an intervening triazole ring, as potential inhibitors of P-gp mediated efflux. Calcein-AM substrate accumulation assays were performed in an MCF7/DX1 cell line that overexpresses P-gp to monitor the inhibitory activity of the clicked quinine dimers. A small library of potent P-gp inhibitors with varying tether lengths is reported, with the best dimer demonstrating low micromolar efficacy.

Amide-modified prenylcysteine based Icmt inhibitors: Structure-activity relationships, kinetic analysis and cellular characterization.

Human protein isoprenylcysteine carboxyl methyltransferase (hIcmt) is the enzyme responsible for the α-carboxyl methylation of the C-terminal isoprenylated cysteine of CaaX proteins, including Ras proteins. This specific posttranslational methylation event has been shown to be important for cellular transformation by oncogenic Ras isoforms. This finding led to interest in hIcmt inhibitors as potential anti-cancer agents. Previous analog studies based on N-acetyl-S-farnesylcysteine identified two prenylcysteine-based low micromolar inhibitors (1a and 1b) of hIcmt, each bearing a phenoxyphenyl amide modification. In this study, a focused library of analogs of 1a and 1b was synthesized and screened versus hIcmt, delineating structural features important for inhibition. Kinetic characterization of the most potent analogs 1a and 1b established that both inhibitors exhibited mixed-mode inhibition and that the competitive component predominated. Using the Cheng-Prusoff method, the K(i) values were determined from the IC(50) values. Analog 1a has a K(IC) of 1.4±0.2µM and a K(IU) of 4.8±0.5µM while 1b has a K(IC) of 0.5±0.07µM and a K(IU) of 1.9±0.2µM. Cellular evaluation of 1b revealed that it alters the subcellular localization of GFP-KRas, and also inhibits both Ras activation and Erk phosphorylation in Jurkat cells.

Photoswitchable Isoprenoid Lipids Enable Optical Control of Peptide Lipidation.

Photoswitchable lipids have emerged as attractive tools for the optical control of lipid bioactivity, metabolism, and biophysical properties. Their design is typically based on the incorporation of an azobenzene photoswitch into the hydrophobic lipid tail, which can be switched between its trans- and cis-form using two different wavelengths of light. While glycero- and sphingolipids have been successfully designed to be photoswitchable, isoprenoid lipids have not yet been investigated. Herein, we describe the development of photoswitchable analogs of an isoprenoid lipid and systematically assess their potential for the optical control of various steps in the isoprenylation processing pathway of CaaX proteins in Saccharomyces cerevisiae. One photoswitchable analog of farnesyl diphosphate (AzoFPP-1) allowed effective optical control of substrate prenylation by farnesyltransferase. The subsequent steps of isoprenylation processing (proteolysis by either Ste24 or Rce1 and carboxyl methylation by Ste14) were less affected by photoisomerization of the group introduced into the lipid moiety of the substrate a-factor, a mating pheromone from yeast. We assessed both proteolysis and methylation of the a-factor analogs in vitro and the bioactivity of a fully processed a-factor analog containing the photoswitch, exogenously added to cognate yeast cells. Combined, these data describe the first successful conversion of an isoprenoid lipid into a photolipid and suggest the utility of this approach for the optical control of protein prenylation.

Functional oligomerization of the Saccharomyces cerevisiae isoprenylcysteine carboxyl methyltransferase, Ste14p.

The isoprenylcysteine carboxyl methyltransferase (Icmt) from Saccharomyces cerevisiae, also designated Ste14p, is a 26-kDa integral membrane protein that contains six transmembrane spanning segments. This protein is localized to the endoplasmic reticulum membrane where it performs the methylation step of the CAAX post-translational processing pathway. Sequence analysis reveals a putative GXXXG dimerization motif located in transmembrane 1 of Ste14p, but it is not known whether Ste14p forms or functions as a dimer or higher order oligomer. We determined that Ste14p predominantly formed a homodimer in the presence of the cross-linking agent, bis-sulfosuccinimidyl suberate. Wild-type untagged Ste14p also co-immunoprecipitated and co-purified with N-terminal-tagged His(10)-myc(3)-Ste14p (His-Ste14p). Furthermore, enzymatically inactive His-Ste14p variants L81F and E213Q both exerted a dominant-negative effect on methyltransferase activity when co-expressed and co-purified with untagged wild-type Ste14p. Together, these data, although indirect, suggest that Ste14p forms and functions as a homodimer or perhaps a higher oligomeric species.

Probing the isoprenylcysteine carboxyl methyltransferase (Icmt) binding pocket: sulfonamide modified farnesyl cysteine (SMFC) analogs as Icmt inhibitors.

Human isoprenylcysteine carboxyl methyltransferase (hIcmt) is a promising anticancer target as it is important for the post-translational modification of oncogenic Ras proteins. We herein report the synthesis and biochemical activity of 41 farnesyl-cysteine based analogs versus hIcmt. We have demonstrated that the amide linkage of a hIcmt substrate can be replaced by a sulfonamide bond to achieve hIcmt inhibition. The most potent sulfonamide-modified farnesyl cysteine analog was 6ag with an IC(50) of 8.8±0.5 µM for hIcmt.

Lipid and sulfur substituted prenylcysteine analogs as human Icmt inhibitors.

Inhibition of isoprenylcysteine carboxyl methyltransferase (Icmt) offers a promising strategy for K-Ras driven cancers. We describe the synthesis and inhibitory activity of substrate-based analogs derived from several novel scaffolds. Modifications of both the prenyl group and thioether of N-acetyl-S-farnesyl-L-cysteine (AFC), a substrate for human Icmt (hIcmt), have resulted in low micromolar inhibitors of Icmt and have given insights into the nature of the prenyl binding site of hIcmt.

The roles of the human ATP-binding cassette transporters P-glycoprotein and ABCG2 in multidrug resistance in cancer and at endogenous sites: future opportunities for structure-based drug design of inhibitors.

The ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp) and ABCG2 are multidrug transporters that confer drug resistance to numerous anti-cancer therapeutics in cell culture. These findings initially created great excitement in the medical oncology community, as inhibitors of these transporters held the promise of overcoming clinical multidrug resistance in cancer patients. However, clinical trials of P-gp and ABCG2 inhibitors in combination with cancer chemotherapeutics have not been successful due, in part, to flawed clinical trial designs resulting from an incomplete molecular understanding of the multifactorial basis of multidrug resistance (MDR) in the cancers examined. The field was also stymied by the lack of high-resolution structural information for P-gp and ABCG2 for use in the rational structure-based drug design of inhibitors. Recent advances in structural biology have led to numerous structures of both ABCG2 and P-gp that elucidated more clearly the mechanism of transport and the polyspecific nature of their substrate and inhibitor binding sites. These data should prove useful helpful for developing even more potent and specific inhibitors of both transporters. As such, although possible pharmacokinetic interactions would need to be evaluated, these inhibitors may show greater effectiveness in overcoming ABC-dependent multidrug resistance in combination with chemotherapeutics in carefully selected subsets of cancers. Another perhaps even more compelling use of these inhibitors may be in reversibly inhibiting endogenously expressed P-gp and ABCG2, which serve a protective role at various blood-tissue barriers. Inhibition of these transporters at sanctuary sites such as the brain and gut could lead to increased penetration by chemotherapeutics used to treat brain cancers or other brain disorders and increased oral bioavailability of these agents, respectively.

Potential Tools for Eradicating HIV Reservoirs in the Brain: Development of Trojan Horse Prodrugs for the Inhibition of P-Glycoprotein with Anti-HIV-1 Activity.

Combination antiretroviral therapy is the mainstay of HIV treatment, lowering plasma viral levels below detection. However, eradication of HIV is a major challenge due to cellular and anatomical viral reservoirs that are often protected from treatment by efflux transporters, such as P-glycoprotein (P-gp) at the blood-brain barrier (BBB). Herein we described a Trojan horse approach to therapeutic evasion of P-gp based on a reversibly linked combination of HIV reverse transcriptase and protease inhibitors. Potent inhibition of P-gp efflux in cells, including human brain endothelial cells, was observed with the linked heterodimeric compounds. In vitro regeneration of active monomeric drugs was observed in a reducing environment with these dimeric prodrugs, with the superior leaving group promoting more facile release from the tether. These release trends were mirrored in the efficacy of the in cyto anti-HIV-1 activity of the Trojan horse heterodimers.

Inhibition of P-glycoprotein-mediated paclitaxel resistance by reversibly linked quinine homodimers.

P-glycoprotein (P-gp), an ATP-dependent drug efflux pump, has been implicated in multidrug resistance of several cancers as a result of its overexpression. In this work, rationally designed second-generation P-gp inhibitors are disclosed, based on dimerized versions of the substrates quinine and quinidine. These dimeric agents include reversible tethers with a built-in clearance mechanism. The designed agents were potent inhibitors of rhodamine 123 efflux in cultured cancer cell lines that display high levels of P-gp expression at the cell surface and in transfected cells expressing P-gp. The quinine homodimer Q2, which was tethered by reversible ester bonds, was particularly potent (IC(50) approximately 1.7 microM). Further studies revealed that Q2 inhibited the efflux of a range of fluorescent substrates (rhodamine 123, doxorubicin, mitoxantrone, and BODIPY-FL-prazosin) from MCF-7/DX1 cells. The reversibility of the tether was confirmed in experiments showing that Q2 was readily hydrolyzed by esterases in vitro (t(1/2) approximately 20 h) while demonstrating high resistance to nonenzymatic hydrolysis in cell culture media (t(1/2) approximately 21 days). Specific inhibition of [(125)I]iodoarylazidoprazosin binding to P-gp by Q2 verified that the bivalent agent interacted specifically with the drug binding site(s) of P-gp. Q2 was also an inhibitor of verapamil-stimulated ATPase activity. In addition, low concentrations of Q2 stimulated basal P-gp ATPase levels. Finally, Q2 was shown to inhibit the transport of radiolabeled paclitaxel (Taxol) in MCF-7/DX1 cells, and it completely reversed the P-gp-mediated paclitaxel resistance phenotype.